CN102026971B - Indolesulfonyl protecting groups for protection of guanidino and amino groups - Google Patents

Indolesulfonyl protecting groups for protection of guanidino and amino groups Download PDF

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Publication number
CN102026971B
CN102026971B CN200980118481.3A CN200980118481A CN102026971B CN 102026971 B CN102026971 B CN 102026971B CN 200980118481 A CN200980118481 A CN 200980118481A CN 102026971 B CN102026971 B CN 102026971B
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compound
alkyl
peptide compound
peptide
hydrogen
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CN102026971A (en
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马蒂厄·吉罗
费尔南多·阿尔贝利西欧
阿尔韦特·伊西德罗·略韦特
梅赛德斯·阿尔瓦雷斯·多明戈
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Polypeptide Laboratory Ppl Holding Co
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Lonza AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Indole Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to indolesulfonyl halogenides which are useful for the protection of organic compounds comprising at least one guanidino moiety and/or at least one amino group. The invention further relates to a process for their preparation and their use as protecting reagents. The invention also relates to the process for the protecting reaction and to the protected compounds thereof.

Description

For the protection of the indoles alkylsulfonyl protectiveness group of guanidine radicals and amino group
Technical field
The present invention relates to such compound, it can be used in the organic compound that protection contains at least one guanidine base section and/or at least one amino group.The present invention relates in addition the preparation method of these compounds and they are as protectant purposes.The invention still further relates to method and the compound that relates to its protection for the protection of reaction.
Background technology
The protection that guanidine base section is suitable remains an open question in chemistry, this owing to known protectiveness group be difficult to remove.This is specially adapted to peptide chemistry, because extremely important for the preparation of multi-medicament with the natural amino acid arginine of guanidine base section.In coupling reaction process, must carry out guanidine radicals protection to arginine, avoid potential acidylate, and de-guanidine subsequently, and therefore cause the less desirable ornithine and the δ-lactan that form.
Depend on the strategy of coupling; tolysulfonyl (Tos) for the arginic protectiveness group the most generally using; 2,2,5; 7; 8-pentamethyl-benzo dihydropyrane-6-sulphonyl (Pmc) and 2,2,4; 6,7-pentamethyl-Dihydrobenzofuranes-5-sulphonyl (Pbf).But these protectiveness groups have too high acid acceptance, therefore need the harsh condition of removing and longer removing the time.So known guanidine radicals protectiveness group tends to form by product in the time that they are removed.Concrete problem is the cracking in their cracking in the peptide with multiple arginine residues or the peptide that containing tryptophane.The people such as Carpino (Tetrahedron Letters1993, the 34th the 49th phase of volume, 7829-7832) have compared Pbf protectiveness group and Pmc protectiveness group when for arginine side chain protected.
WO01/57045 discloses three ring sulfanilamide (SN), its be via cumarone-, thionaphthene-and indoles-intermediate obtain.
described the synthetic of heterocyclic sulfonylurea Deng people, it for example comprises indoles part (J.Heterocyclic Chem., 1996,33,763-766).
Summary of the invention
A target of the present invention is to provide a kind of compound, and it is easy to protect the guanidine base section of organic compound, and it can be easy to remove.
Above-mentioned target is to realize by the compound of claim 1, and this compound can be prepared by the method for claim 5, and its guard method for claim 12 according to claim 9, and the compound of claim 16 is provided.
On the one hand, the present invention relates to the compound of a kind of formula (II):
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With X be chlorine or bromine.
Here term " C and below, 1-nalkyl " be understood to represent any linearity that contains 1-n carbon atom or the alkyl group of branching.For example term " C 1-6alkyl " comprise such as methyl of group, ethyl, propyl group, sec.-propyl, butyl; isobutyl-, sec-butyl, the tertiary butyl, amyl group, isopentyl (3-methyl butyl); neo-pentyl (2,2-dimethyl propyl), hexyl, isohexyl (4-methyl amyl) etc.
Therefore, term " C 1-nalkoxyl group " represent such group, it is by above-mentioned C 1-nalkyl group and the Sauerstoffatom composition being connected by single covalent linkage.
In the same way, term " C 1-6alkyl sulfide " represent such group, it is by above-mentioned C 1-6alkyl group and the sulphur atom composition being connected by single covalent linkage.
Here and below, term " halogen " represents fluorine, chlorine, bromine and iodine.
Favourable, the present invention relates to the compound of formula (II), wherein R 1, R 2, R 3the same with X definition, it does not comprise 1-skatole-3-SULPHURYL CHLORIDE.
In a preferred embodiment, R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide; R 3hydrogen or halogen; With X be chlorine or bromine.
In a kind of specific embodiment, R 1and R 2the two is all methyl; R 3hydrogen; With X be chlorine, this compound is 1,2-dimethyl indole-3-SULPHURYL CHLORIDE.For convenience's sake, this compound will be abbreviated as MIS-CI.
In another aspect of this invention, the compound of formula (II) is prepared by a kind of like this method, and the method comprises step: the compound of formula (I) or its salt are reacted with oxalyl chloride or oxalyl bromine:
Wherein R 1, R 2and R 3definition the same.
In a preferred embodiment, R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, and R 3hydrogen or halogen.
In the preferred embodiment of one, R 1and R 2the two is all methyl, R 3hydrogen.
In the preparation method of formula (II) compound, the sour form of the compound of formula (I) and salt form can be used as reactant.Can use any salt form of the compound of formula (I).Suitable salt is for example sodium salt, sylvite, calcium salt and pyridinium salt.
In a preferred embodiment, described reaction is to carry out with the pyridinium salt of formula (I) compound.
Preferably oxalyl chloride is used for to described reaction.
For this preparation method, can use the mixture of any suitable solvent or suitable solvent.Suitable solvent is such solvent, and it does not react with reactant or with product, and reactants dissolved is arrived enough degree by it.The example of suitable solvent is methylene dichloride, chloroform, tetracol phenixin, tetrahydrofuran (THF) and Isosorbide-5-Nitrae-dioxs.Preferably use methylene dichloride as solvent.This reaction can also solvent-free carrying out.
Easily, this reaction is to carry out under the existence of DMF.
The organic compound that the compound protection of use formula in another aspect of this invention, (II) is contained at least one guanidine base section and/or at least one amino group:
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With X be chlorine or bromine.
In a preferred embodiment, this organic compound is optional resin-carried peptide compound, its be optional side chain protected and/or in free end protection; R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
Here and below, term " peptide compound " is to explain in defined wide in range mode below.So, any compound that term " peptide compound " one of is understood to represent in classification (a) below-(d):
(a) peptide, that is, and by form the compound that amido linkage produces between an amino acid whose carboxyl and another amino acid whose amino.This amido linkage is typically (the true peptide bond) that between an amino acid whose C-1 and another amino acid whose N-2, form, but also represents in term " peptide compound " scope with the compound of the residue connecting by other amido linkages (different peptide bond).The oligomeric peptide being made up of 2-15 amino-acid residue and the bunching propylhomoserin being made up of about 50 amino-acid residues of 16-are the typical peptides of this kind.This amino-acid residue can be any natural or non-natural amino acid.An example of peptide is H-Arg-Val-OH.
(b) amino acid, it can be not only the amino acid (natural a-amino acid) of conventionally finding in protein, and can be any non-natural amino acid.Example is H-Ala-OH (natural amino acid) and homoarginine (alpha-non-natural amino acid).
(c) peptide derivative, represents such peptide, and one or more amino-acid residues are for example by acidylate therein, and alkylation, forms ester or form acid amides and carried out chemical modification.Example is Ac-Phe-Arg-Gly-Ala-Val-OH (SEQ ID NO5), H-Phe-Arg-Gly-Ala-Val-NH 2(SEQ ID NO6) and H-Arg-Gly-Ala-Gly-Gly-Lys (N ε-tetradecanoyl)-Ala-Gly-Gly-OH (SEQ ID NO7).
(d) amino acid derivative, represents such amino acid, and it is for example by acidylate, and alkylation, forms ester or form acid amides and carried out chemical modification.Example is H-AIa-OMe.
In another preferred embodiment, this peptide compound comprises at least one guanidine base section and at least one optional amino group, and this guanidine base section is arginine, homoarginine or go the part of arginine residues.More preferably, described guanidine base section is a part for arginine or homoarginine residue.Even preferred, this peptide compound is Z-Arg-OH.
The prefix " height " (for example homoarginine) of common amino acid name represents that described amino acid comprises an other methylene group in carbochain.
On the contrary, the prefix of common amino acid name " goes " (removing arginine) to represent to have removed a methylene group in carbochain.
In another preferred embodiment, this peptide compound comprises at least one amino group and at least one optional guanidine base section, and at least one described amino group is a part for amino group or the amino-acid residue side chain of N-termination.
Preferred, amino group to be protected is the amino group of the N-termination of described peptide compound.Most preferably this peptide compound is H-Ala-OMe.
Preferred equally, amino group to be protected is a part for the amino-acid residue side chain of described peptide compound.Even preferred, described amino group is Methionin, high-lysine or a part of removing lysine residue.Most preferably, described peptide compound is Z-Lys-OH.
On the other hand, the present invention relates to a kind of method of protecting organic compound, this compound comprises at least one guanidine base section and/or an amino group, and described method comprises reacts described compound with the compound of formula (II):
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With X be chlorine or bromine, therefore so a kind of compound is provided, at least a portion of its contained (III),
Wherein R 1, R 2and R 3as above, m is 0 or 1 in definition.
In one embodiment, m is 1, and therefore so a kind of compound is provided, at least a portion of its contained (IV),
Wherein R 1, R 2and R 3definition as above.
In another embodiment, m is 0, and therefore so a kind of compound is provided, at least a portion that it comprises formula V,
Wherein R 1, R 2and R 3definition as above.
In a preferred embodiment, R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.In one embodiment, m is 1, and in another embodiment, m is 0.
In the preferred embodiment of one, R 1and R 2methyl, R 3be hydrogen, X is chlorine.In one embodiment, m is 1; In another embodiment, m is 0.
In another preferred embodiment; this organic compound is optional resin-carried peptide compound, its be optional side chain protected and/or free end protection, therefore described peptide compound be provided; the at least a portion of its contained (III)
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; M is 0 or 1.
Preferably R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide; R 3hydrogen or halogen.Most preferably R 1and R 2it is methyl; R 3hydrogen.
In a preferred embodiment, described peptide compound comprises at least one guanidine base section, this guanidine base section is arginine, homoarginine or go the part of arginine residues, a preferably part for arginine or homoarginine residue, therefore provide described peptide compound, at least a portion of its contained (III), wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; M is 1; Preferably R wherein 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide; R 3hydrogen or halogen; More preferably R wherein 1and R 2it is methyl; R 3hydrogen.
Most preferably the peptide compound of described question response only comprises a guanidine base section.
In a preferred embodiment, the peptide compound of described question response is N-α-benzyloxy-carbonyl-L-arginine (Z-Arg-OH).
In the even preferred embodiment of one, the peptide compound of question response is Z-Arg-OH; The R of formula (II) compound 1and R 2methyl, the R of formula (II) compound 3be hydrogen, the X of formula (II) compound is chlorine, and therefore N-α-benzyloxycarbonyl-N-ω-(1,2-dimethyl indole-3-alkylsulfonyl)-L-arginine is provided.For convenient, this compound is abbreviated as Z-Arg (MIS)-OH below.
In another preferred embodiment, described peptide compound comprises at least one amino group, at least one described amino group is a part for amino group or the amino-acid residue side chain of N-termination, therefore described peptide compound is provided, the at least a portion of its contained (III), wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With m be 0; Preferably R wherein 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide; R 3hydrogen or halogen; More preferably, R wherein 1and R 2methyl, R 3hydrogen.
Most preferably, the peptide compound of described question response only comprises an amino group, and it is that the amino group of N-termination or its are parts for amino-acid residue side chain.
Even preferred, the amino group of the peptide compound of described question response is the amino group of N-termination.
In a preferred embodiment, the peptide compound of described question response is L-Beta Alanine methyl esters (H-Ala-OMe).
In the even preferred embodiment of one, this peptide compound is H-Ala-OMe; The R of formula (II) compound 1and R 2methyl, the R of formula (II) compound 3be hydrogen, the X of formula (II) compound is chlorine, and therefore N-α-(1,2-dimethyl indole-3-alkylsulfonyl)-L-Beta Alanine methyl esters is provided.For convenient, this compound is abbreviated as MIS-AIa-OMe below.
Equally even preferred, the amino group of the peptide compound of described question response is a part for amino-acid residue side chain.Most preferably described amino group is Methionin, high-lysine or a part of removing lysine residue.
As the solvent for this guard method, can use any inert liq solvent that can solubilizing reaction thing.Operable solvent comprises such as methylene dichloride of halon, tetracol phenixin and ethylene dichloride; Ethers is Anaesthetie Ether such as, tetrahydrofuran (THF), 2-methyltetrahydrofuran; Such as ethyl acetate of carboxyl ester and lactone, methyl acetate and valerolactone; With contain heteroatomic organic solvent such as acetone, acetonitrile, dimethyl formamide and dimethyl sulfoxide (DMSO).This solvent can use separately or use as mixture.Optional, if the dissolving of reactant needs water to exist, this solvent or solvent mixture can comprise water.Preferred solvent is independent or has methylene dichloride and the acetone of water existence.
Optional, this reaction mixture can comprise inorganic or organic bases.The example of mineral alkali is sodium hydroxide, potassium hydroxide, lithium hydroxide and sodium carbonate.The example of organic bases is diisopropyl ethyl amine, pyridine and triethylamine.Preferred alkali is sodium hydroxide and diisopropyl ethyl amine.
The amount of formula (II) compound changes along with reactor volume, and the mol ratio with respect to organic compound (this organic compound comprises at least one guanidine base section and/or an amino group) is 0.9:1-4:1, preferably 1:1-3:1.The compound of formula (II) can merotomize and join in reaction mixture.
This guard method can be carried out at low temperature or slightly high temperature.For example suitable temperature range is-10 DEG C to 30 DEG C, and preferably 0 DEG C is arrived room temperature.
Reaction times is depended on different factors, the mol ratio of the compound of for example temperature or formula (II) and organic compound (this organic compound comprises at least one guanidine base section and/or an amino group).So this reaction can complete at several minutes or in several hours.
On the other hand, the present invention relates to a kind of organic compound, at least a portion of its contained (III),
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; M is 0 or 1.
In one embodiment, m is 1, therefore at least a portion of this organic compound contained (IV):
Wherein R 1, R 2and R 3definition as above.
In another embodiment, m is 0, at least a portion that therefore this organic compound comprises formula V:
Wherein R 1, R 2and R 3definition as above.
In a preferred embodiment, R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.In one embodiment, m is 1, and in another embodiment, m is 0.
In the preferred embodiment of one, R 1and R 2methyl, R 3be hydrogen, X is chlorine.In one embodiment, m is 1, and in another embodiment, m is 0.
In another preferred embodiment, this organic compound is optional resin-carried peptide compound, its be optional side chain protected and/or in free end protection, and at least a portion of its contained (III),
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; M is 0 or 1, and preferably m is 1.
Preferably R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.Most preferably R 1and R 2methyl, R 3hydrogen.
In a preferred embodiment, described peptide compound comprises at least one guanidine base section, this guanidine base section is arginine, homoarginine or go the part of arginine residues, a preferably part for arginine or homoarginine residue, at least a portion of this compound contained (III), wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With m be 1; Preferably R wherein 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide; R 3hydrogen or halogen; More preferably R wherein 1and R 2it is methyl; R 3hydrogen.
Most preferably described peptide compound only comprises a guanidine base section.
Even more preferably described peptide compound is Z-Arg (MIS)-OH.
In another preferred embodiment, described peptide compound comprises at least one amino group, at least one described amino group is a part for amino group or the amino-acid residue side chain of N-termination, at least a portion of its contained (III), wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With m be 0; Preferably R wherein 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide; R 3hydrogen or halogen; More preferably, R wherein 1and R 2methyl, R 3hydrogen.
Preferred equally, described peptide compound only comprises an amino group, and it is that the amino group of N-termination or its are parts for amino-acid residue side chain.
Even preferred, the amino group of described peptide compound is the amino group of N-termination.
Most preferred, described peptide compound is MIS-Ala-OMe.
Equally even preferred, the amino group of described peptide compound is a part for amino-acid residue side chain.Most preferably described amino group is Methionin, high-lysine or a part of removing lysine residue.
Another aspect of the present invention is the organic compound that in the step below, modification and/or coupling obtain according to the present invention.
Because the organic compound obtaining according to the present invention is important member, therefore can be formed with organic compounds with them, this compound can be used as for example drug substance.
According to the present invention, this organic compound is chemical modification in step below, at least a portion of this compound contained (III),
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With m be 0 or 1.
In a preferred embodiment; in step below, the organic compound of modification is optional resin-carried peptide compound; its be optional side chain protected and/or in free end protection, and at least a portion of its contained (III), wherein R 1, R 2and R 3the same with m definition.
Preferably described peptide compound comprises at least one guanidine base section, this guanidine base section is arginine, homoarginine or go the part of arginine residues, a preferably part for arginine or homoarginine residue, at least a portion of this compound contained (III), wherein R 1, R 2and R 3the same with m definition; Preferably R wherein 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen; More preferably R wherein 1and R 2it is methyl; R 3hydrogen.
Most preferably described peptide compound only comprises a guanidine base section.
Even more preferably described peptide compound is Z-Arg (MIS)-OH.
Modification comprises any such organic reaction, and itself and protectiveness group of the present invention adapt.As an example, Z-Arg (MIS)-OH can carry out modification by the deprotection of Z group, has therefore formed H-Arg (MIS)-OH.
Optional, the modified compound therefore obtaining carries out further modification at least one times.As an example, H-Arg (the MIS)-OH obtaining by modification first can carry out further modification by the N-end group of protecting it, has therefore formed for example Fmoc-Arg (MIS)-OH.
This deprotection and protection step can be carried out with the known reaction conditions in the synthetic field of peptide.
According to the present invention, the step by organic compound is below coupled on organic compound Q equally, at least a portion of the former organic compound contained (III):
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With m be 0 or 1.
Optional, described coupling step can repeat at least one times and/or can carry out at least one other coupling with suitable coupling compound.Therefore the compound obtaining can be chemical modification, and can separate from this resin subsequently in described compound is resin-carried situation.
In a preferred embodiment; being somebody's turn to do the organic compound being coupled on organic compound Q in the step is below optional resin-carried peptide compound; its be optional side chain protected and/or in free end protection; the at least a portion of its contained (III), wherein R 1, R 2and R 3the same with m definition.
Preferably described peptide compound comprises at least one guanidine base section, this guanidine base section is arginine, homoarginine or go the part of arginine residues, a preferably part for arginine or homoarginine residue, at least a portion of this compound contained (III), wherein R 1, R 2and R 3the same with m definition; Preferably R wherein 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen; More preferably R wherein 1and R 2it is methyl; R 3hydrogen.
Most preferably described peptide compound only comprises a guanidine base section.
Even more preferably described peptide compound is Fmoc-Arg (MIS)-OH or Z-Arg (MIS)-OH.
Preferably optional resin-carried peptide compound of organic compound Q, its be optional side chain protected and/or free end protection.Most preferably organic compound Q is such peptide compound, its be optional side chain protected be optional resin-carried with it.
In a preferred embodiment, described peptide compound is H-Val-resin or H-Trp (Boc)-Ala-Gly-resin, and preferably this resin stems from Sieber amide resins (9-Fmoc-amino-xanthene-3-oxygen base-Merrifield resin).
In the even preferred embodiment of one, the peptide compound for the treatment of coupling is Fmoc-Arg (MIS)-OH, and the peptide compound that described peptide compound is coupled on it is H-Val-NH-xanthene-3-oxygen base-Merrifield resin, therefore provide Fmoc-Arg (MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin.
Preferably described linked reaction in triplicate, therefore provides Fmoc-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO8).Also preferably after the N-end group deprotection of described resin-carried peptide; by Fmoc-Phe-OH coupling, therefore provide Fmoc-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO1).More preferably by this N-end group deprotection and ethanoyl, therefore provide Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO1).Most preferably described resin-carried peptide is separated from resin, therefore Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH is provided 2(SEQ ID NO2).
In the equally even preferred embodiment of one, the peptide compound for the treatment of coupling is Z-Arg (MIS)-OH, the peptide compound that described compound is coupled on it is H-Trp (Boc)-Ala-Gly-NH-xanthene-3-oxygen base-Merrifield resin, and Z-Arg (MIS)-Trp (Boc)-Ala-Gly-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO3) is therefore provided.Preferably described resin-carried peptide is separated from resin, therefore Z-Arg (MIS)-Trp (Boc)-Ala-GIy-NH is provided 2(SEQ ID NO3).
Coupling process on organic compound Q can carry out according to any coupling method well known by persons skilled in the art.Be in the situation of peptide compound at the organic compound for the treatment of coupling, coupling is preferably carried out in solid phase or liquid phase.Most preferred, the optional posterior coupling step front and optional also carries out in solid phase and/or liquid phase.
Term " solid phase " is understood to represent that solid phase peptide synthesizes (SPPS).In SPPS, amino acid or peptide group (optional protects) are incorporated on solid carrier resin.Then, continuous is attached to amino acid or peptide group (optional protects) on carrier loaded peptide, until form interested peptide material.This carrier loaded peptide then typical case cuts down from carrier, and for further processing and/or purifying.In some cases, solid phase synthesis has produced ripe peptide product; In other cases, the peptide (, " peptide intermediate fragments ") separating with carrier is used to the peptide product that preparation is larger, ripe.
Term " solution phase " is understood to represent that solution phase peptide is synthetic.In solution phase peptide is synthetic; two kinds of peptide intermediate fragments (optional protects); or peptide intermediate fragments and reactive amino acid (the two is all optionally protected) are in suitable solvent; conventionally coupling under other reactant exists, this other reactant has promoted efficiency and the quality of this linked reaction.Peptide intermediate fragments is reactive arrangement, and so that the N-end group of a fragment is coupled on the C-end group of another fragment, vice versa.In addition, side chain protected group (it exists in solid phase synthesis) remains in described fragment conventionally in solution phase coupling process, ensures the specific reactivity of described fragment end.These side chain protected groups are not typically removed, until form ripe peptide compound.
Another aspect of the present invention is the separation of organic compound, at least a portion of this compound contained (III):
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; Or R 1and R 2form together formula-(CH 2) n-part, wherein n is the integer of 3-5; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide, phenyl or benzyl; With m be 0 or 1, what described separation was optional carries out under at least one scavenging agent exists.
In a preferred embodiment, separation method is by using acid, preferably uses trifluoroacetic acid (TFA) to carry out.This acid be independent use or as using with the mixture of inert solvent.
An example of suitable inert solvent is methylene dichloride (DCM).The mol ratio of preferred acid and solvent be 1:0 to 1:2, preferably 1:0 is to 1:1.
At after separating, the sulfonyl compound forming can be collected by any suitable scavenging agent.The example of scavenging agent is tri isopropyl silane (TIS), water, dimethyl sulphide, C 1-4alkoxy benzene for example 1,3,5-trimethoxy-benzene (TMB) and C 1-4alkoxyl group phenol for example 3,4-dimethoxy phenol and 3,5-dimethoxy phenol.This scavenging agent can use separately or use as for example water of mixture/TIS.
Preferably by C 1-4alkoxy benzene and C 1-4alkoxyl group phenol is as scavenging agent, because they are that ratio is as the nucleophilic reagent of the lower polarity of water.As a result of, in the easier working method below of the affixture therefore obtaining, from target compound, separate.
In a preferred embodiment, described organic compound to be separated is optional resin-carried peptide compound, its be optional side chain protected and/or in free end protection.Preferably R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide; R 3hydrogen or halogen, most preferably R 1and R 2methyl, R 3hydrogen.
In the preferred embodiment of one, described peptide compound to be separated is Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO1), and therefore Ac-Phe-Arg-Arg-Arg-Arg-Val-NH is provided 2(SEQ ID NO2).Preferably TFA/DCM/TIS/ water is used as separation solution, most preferred mol ratio is 50:45:2.5:2.5.Same preferred, by TFA/DCM/3,4-dimethoxy phenol uses as separation solution, and most preferred mol ratio is 50:40:10.Same preferred, by TFA/DCM/3,5-dimethoxy phenol uses as separation solution, and most preferably mol ratio is 50:40:10.Same preferred, TFA/DCM/TMB is used as separation solution, most preferred mol ratio is 50:40:10.
In the same preferred embodiment of one, described peptide compound to be separated is Z-Arg (MIS)-Trp (Boc)-Ala-Gly-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO3), and therefore Z-Arg-Trp-Ala-Gly-NH is provided 2(SEQ ID NO4).Preferably TFA/DCM/TMB is used as to separation solution, most preferred mol ratio 50:40:10.
In the same preferred embodiment of one, described peptide compound to be separated is MIS-Ala-OMe, and therefore H-Ala-OMe is provided.Preferably TFA/ dimethyl sulphide is used as to separation solution, most preferred mol ratio is 90:10.
Embodiment
The following examples are further explained the present invention, but object limits absolutely not.Embodiment 1-8 relates to preparation method, and embodiment 9-20 relates to and removes inspection.
If there is no other instructions, use the L-enantiomorph of amino-acid residue, and whole reagent is commercially available.
abbreviation:
Boc=tert-butoxycarbonyl
DIC=di-isopropyl carbon imide
DIPEA=diisopropyl ethyl amine
ESMS=electrospray ionization mass spectrum
Fmoc=fluorenes-9-ylmethoxy carbonyl
HOAt=N-hydroxyl-7-azepine benzotriazole
HOBt=N-hydroxybenzotriazole
HRMS (Cl)=high-res mass spectrum (chemi-ionization)
The substance assistant laser desorpted flight ionization time of MALDI-TOF=
MIS=1,2-dimethyl indole-3-alkylsulfonyl
PyBOP=benzotriazole-1-oxygen base-tripyrrole Wan Tong Phosphonium hexafluorophosphate
TFA=trifluoroacetic acid
TIS=tri isopropyl silane
TMB=1,3,5-trimethoxy-benzene
Z-OSu=N-(benzyloxy carbonyl oxygen base) succinimide
Embodiment 1: pyridine 1, the preparation of 2-dimethyl indole-3-sulfonate
1,2-dimethyl indole (19.7g, 135.9mmol) and sulfur trioxide pyridine complex (20.4g, 128.3mmol) are dissolved in pyridine (100mL) under argon atmospher.By this reaction mixture refluxed 40 hours, then cool to room temperature.Add water after (400mL), formed solution is cleaned 4 times with Anaesthetie Ether (each 250mL).Evaporation water is dried, and in vacuum drier, is dried to produce the pyridine 1 as reddish oil, 2-dimethyl indole-3-sulfonate (37.6g, 96% productive rate).
1H?NMR(400MHz，D 2O)：δ=8.44(d，2H，J=5.8Hz)，8.31(m，1H)，7.75(m，2H)，7.67(d，1H，J=7.7Hz)，7.14(d，1H，J=7.4Hz)，7.05(m，2H)，3.38(s，3H)，2.41(s，3H)。
13C?NMR(100MHz，D 2O)：δ=147.0，140.9，139.2，135.6，127.3，124.1，122.0，121.0，119.2，112.8，109.9，29.2，10.4。
HRMS (Cl): m/z, for C 10h 10nO 3s[M-H] +be calculated as 224.0386, be measured as 224.0388.
Embodiment 2:1, the preparation of 2-dimethyl indole-3-SULPHURYL CHLORIDE (MIS-CI)
By the pyridine of embodiment 11,2-dimethyl indole-3-sulfonate (16.4g, 53.7mmol) at nitrogen atmosphere low suspension in anhydrous methylene dichloride (120mL).Cooling this solution in ice bath, and add slowly oxalyl chloride (14mL, 161mmol).Then, under agitation add very slowly DMF (0.5mL).This reaction mixture is stirred other 30 minutes in ice bath, be then placed in room temperature.After 6 hours, this solution is cooling in ice bath.Add other oxalyl chloride (4mL, 46mmol) and DMF (0.4mL), and by this reaction mixture in stirring at room temperature other 15 hours.Add oxalyl chloride (2mL, 23mmol) and stir after 4 hours in addition, this reaction is carried out (measuring by HPLC completely; Before measuring, with the methyl alcohol processing of little decile 20 minutes).
This reaction mixture room temprature evaporation is dry.Add DMF (200mL), then add water (100mL).Stir this mixture and within 5 minutes, remove oxalyl chloride.Then, be separated, water (each 100mL) cleans organic phase 3 times.This organic phase is dry on anhydrous magnesium sulfate, and evaporation drying, this provides as 1 of purple solid, 2-dimethyl indole-3-SULPHURYL CHLORIDE (MIS-CI) (10.2g, 78% productive rate).
1H?NMR(400MHz，DMSO)：δ=7.82(d，1H，J=7.8Hz)，7.36(d，1H，J=8.0Hz)，7.08(m，2H)，7.00(m，2H)，3.63(s，3H)，2.56(s，3H)。
13C?NMR(100MHZ，DMSO)：δ=137.2，135.9，125.5，121.4，120.8，120.1，109.7，30.0，11.3。
HRMS (Cl): m/z, for C 10h 10nO 2s[M-Cl] +be calculated as 208.0426, be measured as 208.0427.
The preparation of embodiment 3:Z-Arg (MIS)-OH
Z-Arg-OH (2g, 6.5mmol) is dissolved in acetone (65mL) and 3N aqueous sodium hydroxide solution (18mL, 54mmol).This reaction is cooling in ice bath, in 10 minutes, add the MIS-Cl (1.59g, 6.5mmol) that is dissolved in the embodiment 2 in acetone (50mL).This reaction mixture is stirred 1 hour at 0 DEG C.Then, add the other MIS-Cl (0.95g, 3.9mmol) in acetone (20mL), stir 90 minutes at 0 DEG C subsequently.Finally, add the MIS-Cl (0.95g.3.9mmol) in acetone (15mL) of last part.This reaction mixture is stirred other 30 minutes at 0 DEG C, in stirring at room temperature other 3 hours, until can not detect MIS-CI by TLC (hexane: ethyl acetate 1:1).The pH of this reaction neutralizes with 10% aqueous citric acid solution.After acetone vacuum-evaporation, add water (100mL), pH is acidified to pH3 with 10% aqueous citric acid solution.Then, by this ethyl acetate for solution (each 100mL) extraction three times.Organic phase is merged, wash 3 times (each 75mL), dry on magnesium sulfate, final evaporation is dry.Obtained crude product is purified twice by column chromatography (methylene dichloride, methyl alcohol, acetic acid).Merge and purify part, solvent removed in vacuo, obtains a kind of oil.In order to precipitate, add the ethyl acetate of minimum, the mixture of methylene dichloride and methyl alcohol, adds hexane subsequently, until no longer observe other precipitation.Decant solvent, use the mixture of methylene dichloride and hexane to clean solid 4 times, finally dry on magnesium sulfate, Z-Arg (the MIS)-OH of acquisition 18% (0.61g).
1H?NMR(400MHz，DMSO)：δ=7.85(d，1H，J=7.6Hz)，7.52(d，1H，J=8.0Hz)，7.43(d，1H，J=8.0Hz)，7.30(m，5H)，7.10(m，2H)，5.01(s，2H)，3.87(m，1H)，3.66(s，3H)，3.0(m，2H)，2.60(s，3H)，1.64(m，1H)，1.49(m，1H)，1.41(m，2H)。
13C?NMR(100MHZ，DMSO)：δ=174.4，157.0，156.8，139.4，137.7，135.9，129.0，128.5，128.4，125.2，122.1，121.1，120.1，110.4，66.1，54.3，40.0，30.2，28.9，26.4，11.4。
HRMS (CI): m/z, for C 24h 30n 5o 6s[M+H] +, be calculated as 516.1911, be measured as 516.1911.
The preparation of embodiment 4:Fmoc-Arg (MIS)-OH
the preparation of 1.H-Arg (MIS)-OH
The under atmospheric pressure hydrogenation of mixture of methyl alcohol (60mL) solution of the Z-Arg available from embodiment 3 (MIS)-OH (486mg, 0.94mmol) and 10%Pd/C (110mg) is spent the night.Still do not carry out when complete (measuring by TLC in this reaction; Methylene dichloride: methyl alcohol: acetic acid, 90:9:1), add 10%Pd/C (100mg), by other this reaction hydrogenation 24 hours, until complete (measuring by TLC).
This reaction mixture is filtered by celite, and evaporation drying, H-Arg (the MIS)-OH of generation 98% (352mg).
1H?NMR(400MHz，DMSO)：δ=7.83(d，1H，J=7.6Hz)，7.47(d，1H，J=8.1Hz)，7.42(d，1H，J=8.1Hz)，7.11(m，2H)，3.65(s，3H)，3.17(m，1H)，3.00(m，2H)，2.60(s，3H)，1.65(m，1H)，1.54(m，1H)，1.42(m，2H)。
the preparation of 2.Fmoc-Arg (MIS)-OH
Fmoc-Cl (84mg, 0.32mmol) is dissolved in to Isosorbide-5-Nitrae-dioxs (0.5mL).Add the solution of sodiumazide (25mg, 0.39mmol) in water (0.4mL), by formed emulsion stirring at room temperature 2 hours.Then, this emulsion is slowly joined to H-Arg (MIS)-OH (136mg obtaining in preceding step, 0.36mmol) at water with in the solution of the 1:1 mixture of diox, the pH of this solution is 9, this is that the aqueous solution by adding 10% sodium carbonate controls.Stir this reaction mixture, pH is remained on to 9 simultaneously.Once pH has stablized, this mixture is stirred and spent the night.Then add water (30mL), this t-butyl methyl ether for mixture (each 20mL) is cleaned 3 times.With the HCl of 1N be 2-3 by aqueous phase as acidified to pH, then use ethyl acetate (30mL) Rapid Extraction 3 times.Merge organic phase, pass through dried over mgso.After evaporation drying, obtain a kind of oil (115mg), be dissolved in the acetone of minimum.Then, adding pH is 9 aqueous sodium carbonate (20mL), cleans this aqueous solution 3 times by t-butyl methyl ether (each 30mL).Therefore it is 2-3 that the aqueous solution obtaining is acidified to pH with the HCl of 1N, then use ethyl acetate (each 20mL) extraction 3 times, by dried over mgso, final evaporation is dry, produces Fmoc-Arg (the MIS)-OH of 34.3% (67.4mg).
1H?NMR(400MHz，DMSO)：δ=7.86(m，3H)，7.70(d，2H，J=7.4Hz)，7.59(d，1H，J=7.9Hz)，7.42(d，1H，J=8.1Hz)，7.39(m，2H)，7.30(m，2H)，7.10(m，2H)，4.27(m，2H)，4.20(m，1H)13.86(m，1H)，3.66(s，3H)，3.01(m，2H)，2.61(s，3H)，1.65(m，1H)，1.52(m.1H)，1.38(m，2H)。
13C?NMR(100MHZ，DMSO)：δ=174.4，157.0，156.8，144.5，141.4，139.4，135.9，128.3，127.8，126.0，125.2，122.1，121.1，120.8，120.1，110.4，66.3，55.6，47.3，40.0，30.2，28.8，26.5，11.4。
HRMS (Cl): m/z, for C 31h 34n 5o 6s[M+H] +, be calculated as 604.2224, be measured as 604.2222.
Embodiment 5:AC-PrIe-Arg (MiS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-VaI-NH 2the preparation of (SEQ ID NO2)
Sieber amide resins (25mg, 0.42mmol/g, the amino xanthene-3-oxygen of 9-Fmoc-base-Merrifield resin) is put into 2mL polypropylene syringe, and this syringe is provided with polyethylene filtering table.This resin is swelling with methylene dichloride.Subsequently, clean with methylene dichloride and DMF, Fmoc group is removed to (one time 1 minute, twice 10 minutes) by the piperidines with 2:8 and DMF mixture process.Fmoc-Val-OH (14.3mg, 42.1 μ mol) is used to HOBt (5.7mg, 42.1 μ mol) and the solution coupling of DIC (6.7 μ L, 42.1 μ mol) in DMF 1.5 hours.Remove in a usual manner Fmoc group, by the Fmoc-Arg available from embodiment 4 (MIS)-OH (15.8mg, 26.3 μ mol) use PyBOP (13.7mg, 26.3 μ mol), HOAt (3.6mg, 26.3 μ mol) and the solution coupling of DIPEA (13.4 μ L, 78.9 μ mol) in DMF 90 minutes.By this resin by carrying out ethanoyl with the DMF solution-treated 25min of diacetyl oxide (50 equivalent) and DIPEA (50 equivalent); object is by unreacted amine end-blocking; remove Fmoc group; repeat identical program more than 3 times, be included in and remove the Fmoc ethanoyl of resin before.After last Fmoc removes, by Fmoc-Phe-OH (13.6mg, 35 μ mol) use PyBOP (18.3mg, 35 μ mol), HOAt (4.8mg, 35 μ mol) and the solution coupling 90min of DIPEA (17.9 μ L, 105.2 μ mol) in DMF.Remove Fmoc group, by formed free amine group with above-mentioned same mode ethanoyl.By protected resin-carried peptide Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (the MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin N therefore obtaining; dinethylformamide; methylene dichloride and Anaesthetie Ether clean; vacuum-drying, is then divided into five equal portions.
Target compound by equal portions for the preparation of this embodiment, using other equal portions as parent material, for the inspection of removing of for example embodiment 9.
Therefore, equal portions are swelling with methylene dichloride, with the TFA of 1.5mL, methylene dichloride, the mixture process of TIS and water (2:93:2.5:2.5) 20 minutes, separates protected peptide from resin.Filter this resin, collected solution is diluted with methylene dichloride, by adding DIPEA (80 μ L, the TFA of 1.2 equivalents/equivalent) neutralization.Solvent removed in vacuo.After adding water and acetonitrile, by solution freeze-drying, obtain Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH 2(SEQ ID NO2).
This product characterizes with LC-MS and HRMS (Cl): m/z, and for C 80h 107n 23o 15s 4[M+Na] +, being calculated as 1780.7092, gained is 1780.7152.
Embodiment 6:Z-Arg (MIS)-Trp (Boc)-Ala-Gly-NH 2the preparation of (SEQ ID NO4)
Sieber amide resins (70mg, 0.40mmol/g) is put into 2mL polypropylene syringe, and this syringe is provided with polyethylene filtering table.This resin is swelling with methylene dichloride, clean with methylene dichloride and DMF, Fmoc group is removed.By Fmoc-Gly-OH (33.3mg, 112 μ mol), Fmoc-Ala-OH (34.9mg, 112 μ mol) and Fmoc-Trp (Boc)-OH (59.0mg, 112 μ mol) use successively PyBOP (58.3mg, 112 μ mol), HOAt (15.2mg, 112 μ mol) and DIPEA (57.4 μ L, 336 μ mol) solution coupling in DMF 1.5 hours.This resin is divided into 2 equal portions.The a target compound for the preparation of this embodiment, another part is for the preparation of Z-Arg (Pbf)-Trp (Boc)-Ala-Gly-NH 2(referring to embodiment 8.2).
Therefore, by Z-Arg (MIS)-OH (28.9mg, 56 μ mol) with a resin part, use PyBOP (29.2mg, 56 μ mol), HOAt (7.6mg, 56 μ mol) and DIPEA (28.7 μ L, 168 μ mol) solution coupling in DMF 1.5 hours.By protected resin-carried peptide Z-Arg (MIS)-Trp (the Boc)-Ala-Gly-NH-xanthene-3-oxygen base-Merrifield resin N therefore obtaining; dinethylformamide; methylene dichloride and Anaesthetie Ether clean; vacuum-drying, is then divided into the equal portions of 4mg.One equal portions are for the preparation of the target compound of this embodiment, and other equal portions are as parent material, for the inspection of removing of for example embodiment 19.
Therefore, equal portions are swelling with methylene dichloride, with the TFA of 1.5mL, methylene dichloride, the mixture process of TIS and water (2:93:2.5:2.5) 20 minutes, separates protected peptide from resin.Filter this resin, collected solution is diluted with methylene dichloride, by adding DIPEA (80 μ L, the TFA of 1.2 equivalents/equivalent) neutralization.Solvent removed in vacuo.After adding water and acetonitrile, by solution freeze-drying, obtaining purity is Z-Arg (MIS)-Trp (the Boc)-Ala-Gly-NH of 95% (analyzing by HPLC) 2.Obtained product is characterized by LC-MS.
The preparation of embodiment 7:MIS-Ala-OMe
H-Ala-OMe (95mg, 0.68mmol, 1 equivalent) is dissolved in anhydrous methylene chloride, and adds DIPEA (3 equivalent).Add MIS-Cl (200mg, 1.2 equivalents) available from embodiment 2 solution in anhydrous methylene chloride, and by this reaction mixture stirring at room temperature 1.5 hours.Processing in a usual manner, the MIS-Ala-OMe of generation 85.4mg (40%).
The preparation of the control compound of embodiment 8:Pbf protection
8.1
ac-Phe-Arg (Pbf)-Arg (Pbf)-Arg (Pbf)-Arg (Pbf)-Val-NH 2 (SEQ ID? nO2) preparation
Use and preparation Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH 2identical program (referring to embodiment 5), except replacing outside Fmoc-Arg (MIS)-OH with Fmoc-Arg (Pbf)-OH (17.1mg, 26.3 μ mol).Obtained product is characterized by LC-MS and HRMS (Cl): m/z, for C 92h 136n 19o 19s 4[M+H] +calculating is 1938.9137, is measured as 1938.9202.
8.2Z-Arg (Pbf)-Trp (Boc)-Ala-Glv-NH 2 the preparation of (SEQ ID NO4)
By Fmoc-Arg (Pbf)-OH (36.3mg, 56 μ mol) with another part of resin of embodiment 6, use PyBOP (29.2mg, 56 μ mol), HOAt (7.6mg, 56 μ mol) and the solution coupling of DIPEA (28.7 μ L, 168 μ mol) in DMF 1.5 hours.Remove Fmoc group, by processing with Z-OSu (14.0mg, 56 μ mol) and DIPEA (35.9 μ L, 210 μ mol), use Z radical protection unhindered amina.By protected resin-carried peptide Z-Arg (Pbf)-Trp (the Boc)-Ala-Gly-NH-xanthene-3-oxygen base-Merrifield resin N therefore obtaining; dinethylformamide; methylene dichloride and Anaesthetie Ether clean; vacuum-drying, is then divided into the equal portions of 4mg.
One equal portions are for the preparation of the target compound of this embodiment, and other equal portions are as parent material, for the inspection of removing of for example embodiment 20.
Therefore, separate equal portions in identical mode described in embodiment 5.Obtaining purity is Z-Arg (Pbf)-Trp (the Boc)-Ala-Gly-NH of 96% (measuring by HPLC) 2.This product is characterized with LC-MS.
the preparation of 8.3Pbf-Ala-OMe
Be similar to embodiment 7 and prepare, except replacing MIS-CI with Pbf-CI (1.2 equivalent).Productive rate: Pbf-Ala-Ome is 209mg (82%).
Compared with the resin-carried peptide of embodiment 9-12:MIS and Pbf protection, remove inspection
General procedure
Separation solution for protected resin-carried peptide (3mg) (50 μ L) is processed.After disengaging time, this solution is poured in water (4mL).Then, evaporation TFA and methylene dichloride.Formed methylene dichloride for the aqueous solution (each 1mL) is cleaned 6 times, and freeze-drying.Formed solid is analyzed by HPLC (λ=220nm) and ESMS or MALDI-TOF.
Table 1: embodiment 9-12, uses separation solution TFA/DCM/TIS/ water (50:45:2.5:2.5) (water and TIS are as scavenging agent)
A=Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO1), available from embodiment 5.
B=Ac-Phe-Arg (Pbf)-Arg (Pbf)-Arg (Pbf)-Arg (Pbf)-Val-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO1), available from embodiment 8.1 (comparative example).
DCM=methylene dichloride.
Embodiment 13-15: use the MIS protection of different scavenging agents resin-carried peptide remove inspection
General procedure is according to described in embodiment 9-12.Use resin-carried peptide Ac-Phe-Arg (MIS)-Arg (MIS)-Arg (MIS)-Arg (the MIS)-Val-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID NO1) available from the protection of embodiment 5.Disengaging time is 60 minutes.By TFA, the mixture of methylene dichloride and scavenging agent (50:40:10) is as separation solution.
The scavenging agent of testing is 3,4-dimethoxy phenol (embodiment 13), 1,3,5-trimethoxy-benzene (TMB) (embodiment 14) and 3,5-dimethoxy phenol (embodiment 15).
As a result, compare with the embodiment 9 that TIS (2.5%) is used as scavenging agent with water (2.5%) wherein, the amount of MIS-OH has reduced and has been greater than 10 times.In the situation of Tmb, even observe to have reduced and be greater than 40 times.
The resin-carried Tr that contains of embodiment 16 and 17:MIS and Pbf protection ppeptide remove inspection
General procedure is according to described in embodiment 9-12.The crude product forming characterizes by LC-MS, and its purity is analyzed by HPLC (λ=220nm).
The parent material that the purity of the crude product forming is protected higher than Pbf for the parent material of MIS protection.For protected peptide c and d the two, in formed product, do not observe less desirable Trp alkylation, do not observe sulfonation yet.
Table 2: embodiment 16 and 17; Use separation solution TFA/DCM/TMB (50:40:10) (TMB is as scavenging agent)
C=Z-Arg (MIS)-Trp (Boc)-Ala-Gly-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID
NO3), available from embodiment 6.
D=Z-Arg (Pbf)-Trp (Boc)-Ala-Gly-NH-xanthene-3-oxygen base-Merrifield resin (SEQ ID
NO3), available from embodiment 8.2 (comparative example).
DCM=methylene dichloride-TMB=1,3,5-trimethoxy-benzene-* Z-Arg (MIS)-Trp-Ala-Gly-NH 2(SEQ ID NO4) do not detect Z-Arg (Pbf)-Trp-Ala-Gly-NH of (passing through LC-MS)-* * 20.4% in formed crude product 2(SEQ ID NO4) detects (passing through HPLC) in formed crude product.
Embodiment 18-20:MIS and Pbf (as N alpha-amino group protectiveness group) remove inspection
Use separation solution in room temperature treatment in the amino acid of this protection, subsequently by TLC deprotection.
After 5min, MIS removes faster a little, has shown positive Kaiser test (having represented the existence of unhindered amina), and for the situation of Pbf, in ensuing contrast, (10min) is positive Kaiser test.
Table 3: embodiment 18-20; Use separation solution TFA/ methyl-sulfide (90:10) (methyl-sulfide is as scavenging agent)
E=MIS-Ala-OMe, available from embodiment 7.
F=Pbf-Ala-OMe, available from embodiment 8.3 (comparative example).
* the existence that detects H-Ala-OMe by Kaiser does not still exist.

Claims (51)

1. the purposes of the compound of formula (II)
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide; X is chlorine or bromine;
It is as protective material, and for the protection of peptide compound, wherein said peptide compound is any compound one of in classification (a)-(c) below:
(a) natural a-amino acid or homoarginine;
(b) be selected from Ac-Phe-Arg-Gly-Ala-Val-OH (SEQ ID NO5), H-Phe-Arg-Gly-Ala-Val-NH 2(SEQ ID NO6) and H-Arg-Gly-Ala-Gly-Gly-Lys (N ε-tetradecanoyl) peptide of-Ala-Gly-Gly-OH (SEQ ID NO7);
(c)H-AIa-OMe。
2. the purposes of claim 1, wherein this peptide compound is further by resin-carried; R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
3. the purposes of claim 1, wherein this peptide compound be side chain protected and/or protect at free end; R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
4. the purposes of the compound of formula (II)
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide; X is chlorine or bromine;
It is as protective material; for the protection of peptide compound; wherein said peptide compound comprises at least one guanidine base section and at least one amino group alternatively, and wherein said at least one guanidine base section is arginine, homoarginine or a part of going arginine residues.
5. the purposes of claim 4, wherein this peptide compound is further by resin-carried; R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
6. the purposes of claim 4, wherein this peptide compound be side chain protected and/or protect at free end; R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
7. the purposes of claim 4, the part that wherein this guanidine base section is arginine or homoarginine residue.
8. the purposes of claim 4, the part that wherein this guanidine base section is arginine residues.
9. the purposes of the compound of formula (II)
Wherein R 1hydrogen, C 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 2c 1-6alkyl, C 1-6alkoxyl group or C 1-6alkyl sulfide; R 3hydrogen, halogen, C 1-6alkyl, C 1-6alkoxyl group, C 1-6alkyl sulfide; X is chlorine or bromine;
It is as protective material; for the protection of peptide compound; wherein said peptide compound comprises at least one amino group and at least one guanidine base section alternatively, and wherein said at least one amino group is N-end amino group or Methionin, high-lysine or a part of removing lysine side-chain.
10. the purposes of claim 9, wherein this peptide compound is further by resin-carried; R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
The purposes of 11. claims 9, wherein this peptide compound be side chain protected and/or protect at free end; R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
The purposes of 12. claims 9, wherein this amino group is the amino group of N-end amino group or lysine side-chain.
The purposes of 13. claims 9, wherein this amino group is N-end amino group.
Protect the method for peptide compound for 14. 1 kinds; this peptide compound is that claim 1 is defined; the method comprises step: described peptide compound is reacted with the compound of formula (II); the compound of this formula (II) is that claim 1 is defined; therefore so a kind of compound is provided; the at least a portion of its contained (III)
Wherein R 1, R 2, R 3definition with claim 1, m is 0 or 1.
The method of 15. claims 14, wherein R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
The method of 16. claims 14 or 15, wherein R 1and R 2methyl, R 3be hydrogen, X is chlorine.
The method of 17. claims 14 or 15, wherein this peptide compound is further by resin-carried.
The method of 18. claims 16, wherein this peptide compound is further by resin-carried.
The method of 19. claims 14 or 15, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 20. claims 16, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 21. claims 17, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 22. claims 18, wherein this peptide compound be side chain protected and/or protect at free end.
Protect the method for peptide compound for 23. 1 kinds; this peptide compound is that claim 4 is defined; the method comprises step: described peptide compound is reacted with the compound of formula (II); the compound of this formula (II) is that claim 4 is defined; therefore so a kind of compound is provided; the at least a portion of its contained (III)
Wherein R 1, R 2, R 3definition with claim 4, m is 0 or 1.
The method of 24. claims 23, wherein R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
The method of 25. claims 23 or 24, wherein R 1and R 2methyl, R 3be hydrogen, X is chlorine.
The method of 26. claims 23 or 24, wherein this peptide compound is further by resin-carried.
The method of 27. claims 25, wherein this peptide compound is further by resin-carried.
The method of 28. claims 23 or 24, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 29. claims 25, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 30. claims 26, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 31. claims 27, wherein this peptide compound be side chain protected and/or protect at free end.
Protect the method for peptide compound for 32. 1 kinds; this peptide compound is that claim 9 is defined; the method comprises step: described peptide compound is reacted with the compound of formula (II); the compound of this formula (II) is that claim 9 is defined; therefore so a kind of compound is provided; the at least a portion of its contained (III)
Wherein R 1, R 2, R 3definition with claim 9, m is 0 or 1.
The method of 33. claims 32, wherein R 1and R 2independently C 1-4alkyl, C 1-4alkoxyl group or C 1-4alkyl sulfide, R 3hydrogen or halogen.
The method of 34. claims 32 or 33, wherein R 1and R 2methyl, R 3be hydrogen, X is chlorine.
The method of 35. claims 32 or 33, wherein this peptide compound is further by resin-carried.
The method of 36. claims 34, wherein this peptide compound is further by resin-carried.
The method of 37. claims 32 or 33, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 38. claims 34, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 39. claims 35, wherein this peptide compound be side chain protected and/or protect at free end.
The method of 40. claims 36, wherein this peptide compound be side chain protected and/or protect at free end.
41. 1 kinds of peptide compounds, its method by claim 14 obtains.
The compound of 42. claims 41, wherein this peptide compound is further by resin-carried.
The compound of 43. claims 41 or 42, wherein this peptide compound be side chain protected and/or free end protection.
44. 1 kinds of peptide compounds, its method by claim 23 obtains.
The compound of 45. claims 44, wherein this peptide compound is further by resin-carried.
The compound of 46. claims 44 or 45, wherein this peptide compound be side chain protected and/or free end protection.
The compound of 47. claims 44, it is N-α-benzyloxycarbonyl-N-ω-(1,2-dimethyl indole-3-alkylsulfonyl)-L-arginine.
The compound of 48. claims 44, it is N-α-Fmoc-N-ω-(1,2-dimethyl indole-3-alkylsulfonyl)-L-arginine.
49. 1 kinds of peptide compounds, its method by claim 32 obtains.
The compound of 50. claims 49, wherein this peptide compound is further by resin-carried.
The compound of 51. claims 49 or 50, wherein this peptide compound be side chain protected and/or free end protection.
CN200980118481.3A 2008-05-05 2009-05-05 Indolesulfonyl protecting groups for protection of guanidino and amino groups Expired - Fee Related CN102026971B (en)

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